Observing and understanding the activity of a single molecule, such as an enzyme, is critical to understanding the dynamics of many important biochemical processes, such as catalysis, signal transduction, and gene regulation. Many biochemical reactions require micromolar ligand concentrations. In order to perform spectroscopy on one or a few molecules at such high concentrations, it is necessary to limit the size of the effective observation volume.
Previous attempts at sub-diffraction limited spectroscopy have included the utilization of near-field apertures. These implementations typically involve an optical fiber that has been tapered to a sub-wavelength point and coated with a metal such as aluminum. A subwavelength aperture is formed in the metal at the end of the fiber. Excitation light is sent down the fiber towards the aperture, and the elements to be studied are present outside the fiber and in close proximity to the aperture. The subwavelength nature of the aperture results in a light diffraction pattern that includes evanescent modes. These modes rapidly decay with distance from the aperture, thus effectively confining the volume of illumination. Only a very small percentage of light sent down the fiber makes it through the near-field aperture to the illumination region, making the prior art very inefficient.
Additionally, the spectroscopic signal from the analyte is best collected externally by an additional apparatus, such as a microscope objective, since the efficiency of collection by the near-field fiber is very low.
The present invention is directed to overcoming these deficiencies in the prior art.